Anti-cancer activity of (E)-1-(3′,4′-dimethoxyphenyl) butadiene

ABSTRACT

The present invention provides a method of using (E)-1-(3′, 4′-dimethoxyphenyl) butadiene to induce anti-tumorigenic effects in pancreatic carcinoma, ovarian carcinoma, prostate carcinoma, colon carcinoma, lung carcinoma, lymphoblastoma, melanoma and colorectal carcinoma. The invention also provides a method of using (E)-1-(3′, 4′-dimethoxyphenyl) butadiene to prevent cancer metastasis.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

The present invention is a continuation-in-part patent application ofU.S. Ser. No. 15/361,053 filed 24 Nov. 2016 which in turn is anon-provisional filing of provisional patent application 62/275,847filed on 7 Jan. 2016. U.S. Ser. No. 15/361,053 incorporated herein belowfor reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The invention in general relates to cancer therapeutics. Morespecifically, the present invention relates to the ability of (E)-1-(3′,4′-Dimethoxyphenyl) butadiene [DMPBD] to exert anti-tumorigenic andanti-metastatic activity.

Description of Prior Art

It is well known that chronic inflammation increases the risks of cancerand eliminating inflammation reduces the risk for cancer development.Hence anti-inflammatory agents are encouraged to be used in tandem (asadjuvants) with chemotherapeutic agents wherein these anti-inflammatoryagents exert their activity by reducing toxicity of associatedchemotherapy, by altering the pharmacokinetics of chemotherapeuticsresulting in better efficacy and due elimination and also by sensitisingcancerous cells to chemotherapy itself (Elizabeth R. Rayburn et al,“Anti-inflammatory agents for cancer chemotherapy”, Mol Cell Pharmacol.2009; 1 (1): 29-43. It is also known from prior art thatanti-inflammatory substances can also have anti-cancer effects. Forexample, Nonsteroidal anti-inflammatory drugs (NSAIDS) induce apoptosisin various cancer cells. Despite many suggested mechanisms for theanti-cancer effect of NSAIDS including cyclooxygenase inhibition,reactive oxygen species inhibition, and NF-κB mediated signalinhibition, it remains uncertain how they induce cell cycle arrest andapoptosis in cancer cells. In other words, there is little informationon the selectivity of anti-inflammatory agent mediated anti-cancereffects although this information is very critical for successfulanti-cancer therapy and future cancer therapeutic advances (M. Adachi etal, “Nonsteroidal anti-inflammatory drugs and oxidative stress in cancercells”, Histol Histopathol (2007) 22: 437-442). Thus, the prediction ofthe direct effect of such anti-inflammatory molecules in cancer growthor metastasis is difficult (Laurie E Walker, “NSAIDS as anticancerdrugs”, in clinician's brief) and needs considerable scientificevaluation. The anti-inflammatory properties of(E)-1-(3,4-dimethoxyphenyl) butadiene (DMPBD) is well known from priorart (Jeenapongsa et al, “Anti-inflammatory activity of(E)-1-(3,4-dimethoxyphenyl) butadiene from Zingiber cassumunar Roxb.”, JEthnopharmacol. 2003 August; 87(2-3):143-8). Also, the efficacy of thetrans dimer of DMPBD namely, (+/−) trans-3-(3′,4′-dimethoxyphenyl)-4-[(E)-3″′, 4″′-dimethoxy-styryl] cyclohex-1-ene hasbeen discussed in terms of its antiproliferative effects in lung cancer(Journal of Pharmacognosy and Phytochemistry 2015; 4 (1): 01-06).

The present inventors for the first time demonstrate the concentrationdependant in-vitro anti-tumorigenic activity of(E)-1-(3,4-dimethoxyphenyl) butadiene on cancer cell lines PANC-1(pancreatic carcinoma), DU-145 (prostate carcinoma) and SKOV3 (ovariancarcinoma) through cell proliferation assays. Further, the uniqueability of DMPBD to cause significant fold decrease in the expression ofcell adhesion molecules (P-selectin, E-selectin and L-selectin) asevaluated by flow cytometric studies on pancreatic carcinoma cellspoints to the molecule's specific function as an anti-metastatic agent.Important prior art emphasising the role of selectins in tumormetastasis include,

-   a. H. Laubli et al, “Selectins promote tumor metastasis”, Semin    Cancer Biol. 2010 June; 20 (3): 169-77;-   b. Hariri. G et al, “Radiation guided P-selectin antibody targeted    to lung cancer”, Ann Biomed Eng. 2008 May; 36 (5): 821-830;-   c. Chen M et al, “P-selectin mediates adhesion of leucocytes,    platelets and cancer cells in inflammation, thrombosis, cancer    growth and metastasis”, Arch Immunol Ther Exp (Warsz). 2006    March-April; 54 (2): 75-84; and-   d. Okegawa T et al, “The role of cell adhesion molecule in cancer    progression and its application in cancer therapy”, Acta Biochim    Pol. 2004; 51 (2): 445-57.

These scientific developments are important given that cancer therapyobjective depends on

1. Specificity of chemotherapeutic agent to a particular cancer type;and

2. Twin efficacy of said agent in preventing undue cell proliferationand also tumor metastasis.

The inventive features of the present invention as elucidated aboveclearly fulfil the aforesaid objectives by demonstrating theanti-tumorigenic specificity of DMPBD for pancreatic, ovarian andprostate carcinomas and also the ability of DMPBD to prevent spread ormetastasis of these carcinomas or others by inhibition of cell adhesionmolecules P-selectin, E-selectin and L-selectin thus providing novel andnon-obvious technical information useful for chemotherapy of pancreatic,ovarian and prostate cancers and prevention of metastasis thereof.

SUMMARY OF THE INVENTION

The present invention pertains to the anti-tumorigenic andanti-metastatic properties of (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene[DMPBD]. Specifically, the invention discloses the anti-tumorigenicproperties of DMPBD against pancreatic carcinoma, prostate carcinoma andovarian carcinoma. Further, the invention also discloses the ability ofDMPBD to cause significant fold decrease in the expression of celladhesion molecules (P-selectin, E-selectin and L-selectin) as evaluatedby flow cytometric studies on pancreatic carcinoma cells, ovariancarcinoma cells, colon carcinoma cells, lung carcinoma cells,lymphoblastoma cells, prostate carcinoma cells, melanoma cells andcolorectal carcinoma cells. The latter study discloses theanti-metastatic property of DMPBD.

The advantages of the present invention includes the disclosure of theanti-tumorigenic specificity of DMPBD for pancreatic, ovarian, prostate,colon, lung, lymphoblastoma, melanoma and colorectal carcinomas and alsothe ability of DMPBD to prevent spread or metastasis by inhibition ofcell adhesion molecules P-selectin, E-selectin and L-selectin.

Other features and advantages of the present invention will becomeapparent from the following more detailed description, taken inconjunction with the accompanying images, which illustrate, by way ofexample, the principle of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of DMPBD in reducing the expression of adhesionmolecule E-selectin (CD 62E).

FIG. 2 shows the effect of DMPBD in reducing the expression of adhesionmolecule P-selectin (CD 62P).

FIG. 3 shows the effect of DMPBD in reducing the expression of adhesionmolecule L-selectin (CD 62L).

FIG. 4 shows the effect of DMPBD on adhesion molecule P-selectin inProstate carcinoma cell line (LN-CAP)

FIG. 5 shows the effect of DMPBD on adhesion molecule L-selectin inProstate carcinoma cell line (LN-CAP)

FIG. 6 shows the Effect of DMPBD on adhesion molecule P-selectin inLymphoblastoma cell line (K562)

FIG. 7 shows the effect of DMPBD on adhesion molecule L-selectin inLymphoblastoma cell line (K562)

FIG. 8 shows Effect of DMPBD on adhesion molecule P-selectin incolorectal carcinoma cell line (HCT)

FIG. 9 shows effect of DMPBD on adhesion molecule L-selectin incolorectal carcinoma cell line (HCT)

DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS) (FIG. 1, FIG. 2, FIG. 3,FIG. 4, FIG. 5, FIG. 6, FIG. 7, FIG. 8 and FIG. 9)

In the most preferred embodiment, the present invention relates to amethod of inducing anti-tumorigenic effect on human cancer cells, saidmethod comprising step of treating said cancer cells with effectiveamount of (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene. In preferredembodiments, said human cancer cells are selected from group consistingof prostate carcinoma cells, ovarian carcinoma cells, pancreaticcarcinoma cells, colon carcinoma cells, lung carcinoma cells,lymphoblastoma cells, melanoma cells and colorectal carcinoma.

In another most preferred embodiment, the present invention relates to amethod of inhibiting the progression and metastasis of human cancercell, said method comprising step of treating said cancer cell witheffective amount of (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene to bringabout the effect of significant reduction in cell adhesion moleculeexpression thereby inhibiting cancer progression and metastasis. Inpreferred embodiments, the cell adhesion molecule is selected from groupconsisting of P-selectin. E-selectin and L-selectin.

In yet other most preferred embodiments, the present invention relatesto

1. (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene for use in a method oftreating pancreatic carcinoma.

2. (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene for use in a method oftreating ovarian carcinoma.

3. (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene for use in a method oftreating prostate carcinoma.

4. (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene for use in a method oftreating colon carcinoma

5. (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene for use in a method oftreating lung carcinoma

6. (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene for use in a method oftreating lymphoblastoma

7. (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene for use in a method oftreating melanoma

8. (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene for use in a method oftreating colorectal carcinoma

9. (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene for use in treating cancermetastasis wherein (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene arrestsmetastasis by inhibiting the expression of cell adhesion moleculesselected from group consisting of P-selectin, E-selectin and L-selectin.10. Compositions comprising (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene ineffective amounts for use in a method of treating pancreatic carcinoma.11. Compositions comprising (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene ineffective amounts for use in a method of treating ovarian carcinoma.12. Compositions comprising (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene ineffective amounts for use in a method of treating prostate carcinoma.13. Compositions comprising (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene ineffective amounts for use in a method of treating colon carcinoma14. Compositions comprising (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene ineffective amounts for use in a method of treating lung carcinoma15. Compositions comprising (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene ineffective amounts for use in a method of treating lymphoblastoma16. Compositions comprising (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene ineffective amounts for use in a method of treating melanoma17. Compositions comprising (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene ineffective amounts for use in a method of treating colorectal carcinoma18. Compositions comprising (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene ineffective amounts for use in a method of arresting cancer metastasis byinhibiting the expression of cell adhesion molecules selected from groupconsisting of P-selectin, E-selectin and L-selectin.19. Use of (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene in effective amountsfor the manufacture of a medicament for use in treatment of pancreaticcarcinoma.20. Use of (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene in effective amountsfor the manufacture of a medicament for use in treatment of ovariancarcinoma.21. Use of (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene in effective amountsfor the manufacture of a medicament for use in treatment of prostatecarcinoma.22. Use of (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene in effective amountsfor the manufacture of a medicament for use in treatment colon carcinoma23. Use of (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene in effective amountsfor the manufacture of a medicament for use in treatment lung carcinoma24. Use of (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene in effective amountsfor the manufacture of a medicament for use in treatment lymphoblastoma25. Use of (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene in effective amountsfor the manufacture of a medicament for use in treatment melanoma26. Use of (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene in effective amountsfor the manufacture of a medicament for use in treatment colorectalcarcinoma27. Use of (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene in effective amountsfor the manufacture of a medicament for use in arresting cancermetastasis by inhibiting the expression of cell adhesion moleculesselected from group consisting of P-selectin, E-selectin and L-selectin.

Specific illustrative examples enunciating the most preferredembodiments are included herein below.

Example 1: In-Vitro Anti-Tumorigenic Effect of DMPBD on Human CancerCell Lines

Human pancreatic PANC-1 cancer cell line (pancreatic carcinoma cells),human prostate DU145 cancer cell line (prostate carcinoma), humanovarian cancer SKOV3 (ovarian carcinoma) cell line, colon Caco-2 cancercell line (Colon Carcinoma), lung A549 cancer cell line (Lungcarcinoma), lymphoblastoma K562 cancer cell line, melanoma B16F1 cancercell line and colorectal HCT cancer cell line (Colorectal carcinoma),were grown and cultured using standard protocols known in the art. Thecancer cell lines were treated with DMPBD in varying concentrations formeasuring cytotoxicity. The estimation of the viability assay of thecells was done by the MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)colorimetric assay. The MTT Assay measures the cell proliferation rateand conversely, when metabolic events lead to apoptosis or necrosis, thereduction in cell viability. The data is analyzed by plotting cellnumber versus absorbance allowing the quantification of changes in cellproliferation. The rate of tetrazolium reduction is proportional to therate of cell proliferation.

Calculation:

Cytotoxicity of the sample is expressed as IC₅₀ value, the concentrationwhich inhibits 50% of the cell growth.% Cytotoxicity=(E−T/E)×100Where,E=Cell viability in the absence of the sample.T=Cell viability in the presence of the sample

The MTT Assay as performed was adapted from (1) Tim Mosmann. RapidColorimetric Assay for Cellular Growth and Survival: Application toProliferation and Cytotoxicity Assays. Journal of Immunological Methods,65 (1983) 55-63; (2) A. A. van de Loosdrecht, R. H. J. Beelen, G. J.Ossenkoppele, M. G. Broekhoven, M. M. A. C. Langenhuijsen. Atetrazolium-based colorimetric MTT assay to quantitate human monocytemediated cytotoxicity against leukemic cells from cell lines andpatients with acute myeloid leukemia. Journal of Immunological Methods174 (1994) 311-320 and Denis Gerlier and Nicole Thomasset. Use of MTTcolorimetric assay to measure cell activation. Journal of ImmunologicalMethods, 94 (1986) 57-63.

Results

TABLE 1 Serial Number Cell Lines Description IC₅₀ (μg/ml) 1 PANC-1Pancreatic carcinoma 4.2 2 DU 145 Prostate carcinoma 8.6 3 SKOV3 Ovariancarcinoma 13.5 4 Caco-2 Colon Carcinoma 39.3 5 A549 Lung Carcinoma 30.26 K562 Lymphoblastoma 22.5 7 B16F1 Melanoma 26.4 8 HCT ColorectalCarcinoma 25.5

Example 2: Effect of DMPBD on the Expression of Adhesion MoleculesE-Selectin, P-Selectin and L-Selectin (Anti-Metastatic Effect ofDMPBD)—(FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG. 6, FIG. 7, FIG. 8and FIG. 9)

Plating of carcinoma cells was done. The confluent cultures wereharvested by trypsinization and expanded during two more passages beforethey were used for the experiments. Medium and other culture componentswere renewed after 48-72 h. All cell cultures were maintained at 37° C.in 95% air and 5% CO2 in a CO2 incubator. 10 ng/ml of recombinant humanTNF-alpha and IL-1beta was added to the cell culture. DMPBD was added atgraded concentration. After 72 hours the cells were incubated withspecific antibodies PE labeled CD 62E; FITC labeled CD 62P and PElabeled CD 62L in different sets for 5 hours. The cells were thentrypsinised and acquired on Flow cytometer for analyzing the effect ofDMPBD. The Flow cytometric analysis was adapted from the teachings of(1) Curvers J, de Wildt-Eggen J, Heeremans J, Scharenberg J, de Korte D,van der Meer P F. Flow cytometric measurement of CD62P (P-selectin)expression on platelets: a multicenter optimization and standardizationeffort. Transfusion. 2008. 48(7):1439-46, (2) Choi Y W. Kim H J. Park SS. Chung H W. Lee S O. Oh B S. Kim J B. Kim H Y. Chung B P. Yu C D. KimS Y. Inhibition of endothelial cell adhension by the newanti-inflammatory agent alpha-iso-cubebene. Vascul Pharmacol. 2009;51:215-224 and (3) Yen Y T, Liao F, Hsiao C H, Kao C L, Chen Y C,Wu-Hsieh B A. Modeling the early events of severe acute respiratorysyndrome coronavirus infection in vitro. J Virol. 2006 March;80(6):2684-93. The percentage reduction of selectin expression by DMPBDon different cell lines is specified in Table 2.

TABLE 2 Cell DMPBD Serial Line Adhesion Conc. % Number Name Descriptionmolecule (μg/ml) Reduction 1 LNCap Prostate P-selectin 70 11.1 carcinomaL-selectin 20  7.3 2 K562 Lympho- P-selectin 10/20 11.6/35.4 blastomaL-selectin 20 30.3 3 HCT Colorectal P-selectin 10/20 11.8/27.8 CarcinomaL-selectin 10/20 11.3/21.1

DMPBD used in the aforesaid experiments was obtained from the rhizomesof Zingiber cassumunar by proprietary process, the steps of which arehighlighted herein below.

-   -   1. Pulverized material of Z. cassumunar rhizome was extracted        with methanol (4 vol×4) with constant stirring at 60-70° C. The        methanolic extract was pooled and concentrated.    -   2. The concentrate of step 1 was then washed with hexane (4        vol×4) at 55-60° C. and resultant product (hexane extract)        pooled and concentrated;    -   3. The hexane extract of step 2 was then solubilised in methanol        and mixed with γ-cyclodextrin in a ratio of 2:3 with proper        refluxing and vacuum dried at 55-60° C. to yield 90% water        soluble final product with yield 7.79% and assay of DMPBD 10.73%        and assay of DMPB-1-en 0.44%.

While the invention has been described with reference to a preferredembodiment, it is to be clearly understood by those skilled in the artthat the invention is not limited thereto. Rather, the scope of theinvention is to be interpreted only in conjunction with the appendedclaims.

We claim:
 1. A method of inducing anti-tumorigenic effect on coloncarcinoma, lung carcinoma, lymphoblastoma, melanoma and colorectalcarcinoma cells, said method comprising the step of treating said cellswith an effective amount of (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene. 2.A method of inhibiting the progression and metastasis of prostatecarcinoma cells, lymphoblastoma cells and colorectal carcinoma cells,said method comprising the step of treating said prostate carcinomacells, lymphoblastoma cells and colorectal carcinoma cells with aneffective amount of (E)-1-(3′, 4′-Dimethoxyphenyl) butadiene to bringabout the effect of significant reduction in cell adhesion moleculeexpression thereby inhibiting carcinoma progression and metastasiswherein the cell adhesion molecule is selected from the group consistingof P-selectin, E-selectin and L-selectin.